C6orf120 gene knockout in rats mitigates concanavalin A­induced autoimmune hepatitis via regulating NKT cells.

OBJECTIVE: To elucidate the role of the functional unknown gene C6orf120 in the pathogenesis of AIH and its mechanism of action, using C6orf120 knockout rats. METHODS: An autoimmune hepatitis model was established with 35 mg/kg intravenous injection of concanavalin A (Con A) in C6orf120-knockout (C6orf120-/-) and wild-type (WT) rats. Rats were sacrificed after administering Con A for 0, 12, and 24 h. The peripheral blood, liver, spleen, and mesenteric lymph nodes were collected for follow-up studies. RESULTS: C6orf120 knockout significantly decreased the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and improved the histological damage in Con A-induced autoimmune liver injury.Loss of C6orf120 function significantly increased the frequency of CD3+ CD161+ NKT cells in the peripheral blood, liver, and spleen; downregulated the expression of CD314 (NKG2D) in the liver, spleen, and mesenteric lymph nodes; reduced the expression of inflammatory cytokines and chemokines; and suppressed the mRNA and protein expression of Fas and FasL in the liver. Additionally, C6orf120 knockout significantly downregulated the expression of p-JAK1, p-JAK2, p-STAT1, and p-STAT3 in liver tissue. CONCLUSION: The protective effect of C6orf120 knockout against Con A-induced hepatitis may be due to the inhibition of NKT cell activation, restriction of cytokine and chemokine activities, inhibition of JAK-STAT and Fas/FasL signaling pathway activation, and reduction in liver inflammation and hepatocyte apoptosis.

Overexpression of FADD and Bcl-XS proteins as novel prognostic biomarkers for surgically resected non-small cell lung cancer.

BACKGROUND: Non-small cell lung cancer (NSCLC) is one of the most widespread cancer with increasing morbidity and mortality. FAS-associated protein with death domain (FADD) is considered as an essential instrument in cell death, whereas Bcl-XS promotes apoptosis through inhibiting the activity of Bcl-2 and Bcl-XL. OBJECTIVE AND METHODS: We detected the expression of FADD and Bcl-XS in resected NSCLC tissues by immunohistochemistry, and investigated their association with clinicopathological characteristics and prognostic significance of NSCLC patients. RESULTS: Bcl-XS expression was significantly increased in well and moderate differentiated lung SCC (P= 0.004). Lung ADC patients with overexpression of FADD and lung SCC patients with low expression of Bcl-XS had importantly lower overall survival rates by Kaplan-Meier analysis (P= 0.033, P= 0.02, respectively). Multivariate analysis confirmed that elevated expression of FADD was an independent poor prognostic factor for patients with surgically resected lung ADC (P= 0.027) and increased expression of Bcl-XS was an independent good prognostic factor for patients with surgically resected lung SCC (P= 0.016)CONCLUSION: Elevated expression of FADD was identified as independent poor prognostic factor for patients with surgically resected lung ADC, however, increased expression of Bcl-XS was an independent good prognostic biomarker for patients with surgically resected lung SCC.

[Formation of FADD amyloid fiber and its role in immune signaling in Drosophila melanogaster].

In this research, we studied the formation of Drosophila melanogaster FADD (Fas-associated death domain-containing protein) amyloid fiber and its influence on signal transduction in IMD (Immune deficiency) signaling pathway to better understand the regulation mechanism of Drosophila innate immune signaling pathway, which will provide reference for the immune regulation in other species. First, we purified dFADD protein expressed in Escherichia coli and performed Sulfur flavin T binding and transmission electron microscopy to identify the dFADD amyloid fibers formed in vitro. Then we investigated the formation of dFADD polymers in S2 cells using SDD-AGE and confocal microscope. We also constructed dFADD mutants to find out which domain is essential to fiber formation and its effect on IMD signal transduction. Our results revealed that dFADD could be polymerized to form amyloid fiber polymers in vitro and inside the cells. Formation of fibers relies on DED (Death-effector domain) domain of dFADD, since DED domain-deleted mutant existed as a monomer. Dual luciferase reporter assay showed that intact DED domain was required for the induction of downstream antimicrobial peptides, indicating that fiber formation was the key to IMD signal transduction. Our study revealed the role of dFADD in mediating the cascade between IMD and Dredd in the IMD signaling pathway by forming amyloid fibers, suggesting an evolutionarily conserved regulatory mechanism of innate immune signaling pathway.

Topiramate Reverses Physiological and Behavioral Alterations by Postoperative Cognitive Dysfunction in Rat Model Through Inhibiting TNF Signaling Pathway.

This study aimed to investigate the effects of topiramate (TPM) on rats with postoperative cognitive dysfunction (POCD) and elucidate the underlying mechanism. Differentially expressed genes in propofol-treated group and vehicle control group were filtered out and visualized in heatmap based on R program. POCD rat models were established for validation of TPM's anti-inflammatory action and Morris water maze (MWM) test was employed for assessment of spatial learning and memory ability of rats. Hematoxylin and eosin (HE) staining was applied to detect the neurodegeneration, and the apoptosis status was detected using TUNEL assay. In vitro, hippocampal microglia was treated with lipopolysaccharide or TPM to validate the TPM's anti-inflammatory action. Cell apoptosis was detected with flow cytometry. Inflammatory factors were detected by enzyme-linked immunosorbent assay, and factor-associated suicide (Fas), Fas-associated protein with death domain (FADD) expression were detected by western blot. As results, TPM administration improved the spatial learning and memory ability in POCD rat by decreasing the expression levels of Fas, FADD, and inflammatory factors (tumor necrosis factor-α, TNF-α; interleukin-1ß, IL-1ß; interleukin-6, IL-6) in POCD rats. In addition, TPM down-regulated cell apoptotic rate to suppress POCD by decreasing the expression of Caspase8, Bcl2-associated X (Bax), and poly ADP-ribose polymerase-1 (PARP1) yet enhancing B-cell lymphoma-2 (Bcl-2) expression. Besides, inhibition of Fas enhanced TPM-induced down-regulation of apoptosis of neuronal cell in hippocampus tissues of POCD rats. Our results revealed that treatment of POCD rats with TPM could suppress neuronal apoptosis in the hippocampus tissues, and the neuroprotective effects of TPM may relate with the regulation of tumor necrosis factor (TNF) signaling pathway.

Continuous gibberellin A3 exposure from weaning to sexual maturity induces ovarian granulosa cell apoptosis by activating Fas-mediated death receptor signaling pathways and changing methylation patterns on caspase-3 gene promoters.

Information on the effects of gibberellic acid (gibberellin A3, GA3) on ovarian follicle development is limited. In our present study, 21-day-old female Wistar rats were exposed to GA3 by gavage (25, 50, and 100 mg/kg body weight, once per day) for eight weeks to evaluate the influence of GA3 on ovarian follicle development. After treatment, significant (P < 0.05) increases (to 40.17 % and 44.5 %, respectively) in atretic follicle proportions and significant decreases (to 19.49 % and 17.86 %, respectively) in corpus luteum proportions were observed in the 50 and 100 mg/kg treatment groups compared to the control group. Significant (P < 0.05) increases (to 31.3 % and 42.0 %, respectively) in follicle apoptosis were observed in the 50 and 100 mg/kg treatment groups by transmission electron microscopy and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays. Significantly increased expression of caspase-3, caspase-8, caspase-9 and Fas was observed by real-time PCR and Western blotting. Bisulfite sequencing PCR (BSP) revealed obviously decreased total methylation percentages of the caspase-3 promoter region in the two treatment groups. Real-time quantitative PCR also showed significantly decreased mRNA expression of DNA methyltransferase (Dnmt) 3a and Dnmt3b. Further in vitro studies showed that a DNA methylation inhibitor could enhance the GA3-induced increase in the mRNA expression of caspase-3. Overall, our present study indicates that GA3 administration from weaning until sexual maturity can affect ovarian follicle development by inducing apoptosis and suggests that signaling through the Fas-mediated apoptotic pathway may be an important underlying mechanism of this apoptosis. In addition, GA3-induced aberrant DNA methylation patterns might be partly responsible for upregulation of caspase-3 gene expression.

Adenovirus-mediated overexpression FADD induces a significant antitumor effect on human colorectal cancer cells both in vitro and in vivo.

The Wnt/ß-catenin signaling pathway plays important roles in cancers such as colorectal cancer. Colon cancer cells secrete and express high levels of ß-catenin, which may stimulate autocrine signaling and further enhance activities of the canonical Wnt signaling pathway. Free ß-catenin in the cytoplasm and nucleus leads to its association with T cell factor (TCF)/lymphocyte enhancing factor (Lef) transcription factors, and subsequent transcriptional activation of downstream target genes. FADD plays a key role in cellular apoptosis in many different types of cancer. Therefore, a recombinant adenovirus is constructed, in which an apoptosis gene FADD is placed under control of a promoter containing Tcf-responsive elements. It is observed that FADD overexpression can suppress cell growth and enhance apoptosis of SW480 cells in vitro. In addition, Ad-FADD can also suppress the growth of subcutaneous xenografts in the nude mice. Together, these results suggest that Ad-FADD has anti-proliferative and pro-apoptotic effects in colon cancer cells, which provides a novel strategy for treatment of colorectal cancer.

Nitidine chloride inhibits proliferation and induces apoptosis in ovarian cancer cells by activating the Fas signalling pathway.

OBJECTIVES: To explore the apoptotic effects and underlying mechanisms of nitidine chloride (NC) in epithelial ovarian cancer. METHODS: The MTT cell proliferation assay was used to detect the inhibitory effects of different concentrations of NC (0, 0.3125, 0.625, 1.25, 2.5, 5 and 10 µg/ml) in SKOV3 ovarian carcinoma cells. The number of apoptotic cells was observed by Hoechst staining and measured by flow cytometry. Quantitative PCR was used to measure the expression of Fas, Fas-associated death domain-containing protein (FADD), caspase-8 and caspase-3. RNA interference (RNAi) was used to determine whether caspase-8 played an important role in NC-induced apoptosis. KEY FINDINGS: Nitidine chloride inhibited the proliferation of SKOV3 cells (IC50 = 2.317 ± 0.155 µg/ml) after 24 h of treatment and induced apoptosis (15.9-64.3%). Compared with the control group, a significant increase in Fas, FADD, caspase-8 and caspase-3 gene expression was observed in the NC-treated groups (P < 0.05). After silencing caspase-8 by RNAi, the antiproliferative activity and pro-apoptotic activity of NC in SKOV3 cells decreased (P < 0.05). CONCLUSIONS: Our study showed that NC induced apoptosis in SKOV3 cells by activating the Fas signalling pathway, and caspase-8 played an important role in this process.

Decreased cortical FADD protein is associated with clinical dementia and cognitive decline in an elderly community sample.

BACKGROUND: FADD (Fas-associated death domain) adaptor is a crucial protein involved in the induction of cell death but also mediates non-apoptotic actions via a phosphorylated form (p-Ser194-FADD). This study investigated the possible association of FADD forms with age-related neuropathologies, cognitive function, and the odds of dementia in an elderly community sample. METHODS: FADD forms were quantified by western blot analysis in dorsolateral prefrontal cortex (DLPFC) samples from a large cohort of participants in a community-based aging study (Memory and Aging Project, MAP), experiencing no-(NCI, n = 51) or mild-(MCI, n = 42) cognitive impairment, or dementia (n = 57). RESULTS: Cortical FADD was lower in subjects with dementia and lower FADD was associated with a greater load of amyloid-ß pathology, fewer presynaptic terminal markers, poorer cognitive function and increased odds of dementia. Together with the observations of FADD redistribution into tangles and dystrophic neurites within plaques in Alzheimer's disease brains, and its reduction in APP23 mouse cortex, the results suggest this multifunctional protein might participate in the mechanisms linking amyloid and tau pathologies during the course of the illness. CONCLUSIONS: The present data suggests FADD as a putative biomarker for pathological processes associated with the course of clinical dementia.

Tumor-targeted delivery of a C-terminally truncated FADD (N-FADD) significantly suppresses the B16F10 melanoma via enhancing apoptosis.

Fas-associated protein with death domain (FADD), a pivotal adaptor protein transmitting apoptotic signals, is indispensable for the induction of extrinsic apoptosis. However, overexpression of FADD can form large, filamentous aggregates, termed death effector filaments (DEFs) by self-association and initiate apoptosis independent of receptor cross-linking. A mutant of FADD, which is truncated of the C-terminal tail (m-FADD, 182-205 aa) named N-FADD (m-FADD, 1-181 aa), can dramatically up-regulate the strength of FADD self-association and increase apoptosis. In this study, it was found that over-expression of FADD or N-FADD caused apoptosis of B16F10 cells in vitro, even more, N-FADD showed a more potent apoptotic effect than FADD. Meanwhile, Attenuated Salmonella Typhimurium strain VNP20009 was engineered to express FADD or N-FADD under the control of a hypoxia-induced NirB promoter and each named VNP-pN-FADD and VNP-pN-N-FADD. The results showed both VNP-pN-FADD and VNP-pN-N-FADD delayed tumor growth in B16F10 mice model, while VNP-pN-N-FADD suppressed melanoma growth more significantly than VNP-pN-FADD. Additionally, VNP-pN-FADD and VNP-pN-N-FADD induced apoptosis of tumor cells by activating caspase-dependent apoptotic pathway. Our results show that N-FADD is a more potent apoptotic inducer and VNP20009-mediated targeted expression of N-FADD provides a possible cancer gene therapeutic approach for the treatment of melanoma.

Expression of FADD and cFLIPL balances mitochondrial integrity and redox signaling to substantiate apoptotic cell death.

FADD and cFLIP both are pivotal components of death receptor signaling. The cellular signaling of apoptosis accomplished with death receptors and mitochondria follows independent pathways for cell death. FADD and cFLIP both have an important role in the regulation of apoptotic and non-apoptotic functions. Dysregulated expression of FADD and cFLIP is associated with resistance to apoptosis in cancer cells. Mitochondria are known to play critical role in maintaining cellular respiration and homeostasis in the cells as well as transduces various signals to determine the fate of cell death. However, involvement of FADD and cFLIP in regulation of mitochondrial integrity and programmed cell death signaling to define the fate of cells remains elusive. In the present study, we explored that, induced expression of FADD challenges the mitochondrial integrity and pulverizes the membrane potential by altering the expression of Bcl-2 and cytochrome c. In contrast, mutant of FADD was unable to affect the mitochondrial integrity. Interestingly, expression of FADD and cFLIP helps to balance redox potential by regulating the anti-oxidant levels. Further, we noticed that, knockdown of cFLIPL and induced expression of FADD rapidly accumulate intracellular ROS accompanied by JNK1 activation to substantiate apoptosis. Notably, the ectopic expression of cFLIPL resists the sensitivity of cancer cells against apoptosis inducers Etoposide and HA14-1. Altogether, our findings suggest that FADD and cFLIPL are important modulators of mitochondrial-associated apoptosis apart from the death receptor signaling.

Differential activation of CD95-mediated apoptosis related proteins in proximal and distal tubules during rat renal development.

The CD95-mediated apoptotic pathway is the best characterized of the death receptor-mediated apoptotic pathways. The present study characterized localization and expression of proteins involved in CD95-mediated apoptosis during rat renal development. Kidneys were obtained from embryonic (E) 18 and 20-day-old fetuses and postnatal (P) 1-, 3-, 5-, 7-, 14-, and 21-day-old pups. Immunohistochemical characterization revealed that CD95, FasL and cleaved caspase-3 were strongly expressed in proximal tubules and weakly expressed in distal tubules, but that expression of caspase-8 in distal tubules was stronger than that in proximal tubules. Results from terminal deoxynucleotidyl transferase dUTP nick end labeling assays showed that levels of apoptosis in proximal tubules slowly increased after E18, while those of distal tubules slowly decreased after P5. Western blotting demonstrated that expression of CD95, FasL and FADD was very weak during embryonic development, but rapidly increased at P14. Expression of cleaved caspase-3 was maintained at high levels after P1, while caspase-8 expression gradually reached a peak at P7. Results from this study reveal that the CD95-mediated apoptotic pathway is a key driver of apoptosis in proximal tubules during late postnatal kidney development in rats and suggest that apoptosis in distal tubules is mediated by a different apoptotic pathway.

Progesterone augments epirubicin-induced apoptosis in HA22T/VGH cells by increasing oxidative stress and upregulating Fas/FasL.

BACKGROUND: Although epirubicin, an anthracycline drug, is widely used to treat hepatocellular carcinoma, its therapeutic efficacy is disappointing. Thus, the efficacy of epirubicin may be improved when combined with other drugs. This study investigated the therapeutic potential of combination of progesterone and epirubicin in the treatment of the human hepatoma cell line HA22T/VGH and the possible mechanisms through which this combination might induce apoptosis. MATERIALS AND METHODS: HA22T/VGH cells were treated without or with 25 µM progesterone and/or 0.5 µM epirubicin and analyzed for oxidative stress, redox status, Fas/FasL expression, caspase activity, and apoptosis. RESULTS: HA22T/VGH cells treated with epirubicin increased the production of reactive oxygen species and nitric oxide, the expression of Fas, FasL, and Fas-associated death domain, and the activities of caspase-8 and caspase-3. Epirubicin treatment also decreased glutathione resulting in the induction of apoptosis. Treatment with progesterone alone increased nitric oxide production, but it did not affect the other parameters. However, when HA22T/VGH cells were treated with progesterone and epirubicin, the effects of epirubicin were enhanced. CONCLUSIONS: Our observations suggest that progesterone enhances the efficacy of epirubicin. The increased efficacy is potentially attributed to progesterone's enhancement of epirubicin-induced oxidative stress, thereby reducing redox status. In addition, progesterone sequentially upregulates Fas/FasL to induce the caspase-8 and caspase-3 pathways, thereby resulting in increased apoptosis. The combination had a greater effect on the induction of HA22T/VGH cell apoptosis and could potentially serve as a more effective treatment for hepatocellular carcinoma than epirubicin alone.

FADD is upregulated in relapsing remitting multiple sclerosis.

OBJECTIVE: To elucidate the role of tumor necrosis factor (TNF) receptor signal transduction in multiple sclerosis (MS). METHODS: We performed a cross-sectional analysis of the gene expression of TNF receptor-associated death domain protein (TRADD) and Fas-associated death domain protein (FADD) in peripheral blood leukocytes of 23 relapsing remitting (RR), 19 secondary progressive (SP) and 12 primary progressive (PP) MS patients, as well as of 29 healthy controls by quantitative RT-PCR. Additionally, we monitored a subgroup of 15 RR MS patients longitudinally every 3 months over the time period of 9 months. RESULTS: FADD expression was significantly elevated in RR MS patients compared to the other disease courses (p < 0.048). The median of FADD expression was elevated in the RR MS patient groups compared to the healthy group, but this was not significant (p < 0.053). The median of TRADD expression was elevated in the patient groups compared to the healthy group, but this was not significant (p < 0.14). Neither variable changed significantly over the time course of 9 months. CONCLUSION: FADD elevation in leukocytes might be interpreted as the molecular equivalent of an elevated general inflammatory activity in RR MS patients compared to other disease courses. FADD elevation in RR MS reinforces the concept that different pathophysiological and immunological processes sustain RR MS and SP or PP MS.

Induction of apoptosis by diphenyldifluoroketone in osteogenic sarcoma cells is associated with activation of caspases.

The aim of the present study was to investigate and compare the effects of diferuloylmethane (curcumin) and diphenyldifluoroketone (EF-24) on cell growth and apoptosis induction in human osteogenic sarcoma cells. This was examined by MTT assay, nuclear DAPI staining, caspase-activation assay, flow cytometry analysis and immunoblotting in Saos2 human osteogenic sarcoma cells. Curcumin and EF-24 inhibited the growth of Saos2 cells in a dose-dependent manner. The apparent potency of EF-24 was more than 3-fold higher that of curcumin. Treatment with curcumin or EF-24 resulted in nuclear condensation and fragmentation in the cells. The caspase-3/-7 activities were detected in living cells treated with curcumin or EF-24. Flow cytometry showed that the rate of apoptosis was increased by curcumin and EF-24 compared to the control. Curcumin and EF-24 promoted the proteolytic cleavages of procaspase-3/-7/-8/-9 with increases in the amount of cleaved caspase-3/-7/-8/-9. The curcumin- or EF-24-induced apoptosis in the Saos2 cells was mediated by the expression of Fas and activation of caspase-8, caspase-3 and poly(ADP-ribose) polymerase. Immunoblotting revealed the Bid and Bcl-2 proteins to be downregulated, and truncated-Bid, Bax and p53 proteins to be upregulated by curcumin and EF-24. Curcumin and EF-24 increased the Bax/Bcl-2 ratio significantly. These results suggest that the curcumin and EF-24 inhibit cell proliferation and induce apoptotic cell death in Saos2 human osteogenic sarcoma cells via both the mitochondria-mediated intrinsic pathway and the death receptor-mediated extrinsic pathway, and may have potential properties for anti-osteosarcoma drug discovery.

Epigenetic regulation of microRNA-128a expression contributes to the apoptosis-resistance of human T-cell leukaemia jurkat cells by modulating expression of fas-associated protein with death domain (FADD).

Increased expression of miR-128a is often observed in acute lymphoblastic leukaemia (ALL) compared with its expression in acute myeloid leukaemia (AML). The objective of this study was to investigate the role of miR-128a, especially that in the Fas-signalling pathway, in T-cell leukaemia cells. The role of miR-128a in Fas-mediated apoptosis was examined by using Fas-activating antibody (CH-11)-susceptible Jurkat cells and -resistant Jurkat/R cells. Whereas ectopic expression of miR-128a conferred Fas-resistance on Jurkat cells by directly targeting Fas-associated protein with death domain (FADD), antagonizing miR-128a expression sensitized Jurkat/R cells to the Fas-mediated apoptosis through derepression of FADD expression. Myeloid leukaemia HL60 and K562 cells were also CH-11-resistant, sharing a similar resistant mechanism with Jurkat/R cells. Furthermore, CH-11 induced demethylation of the promoter region of miR-128a with resultant up-regulation of miR-128a expression in Jurkat/R cells, which was shown to be a mechanism for the resistance ofJurkat/R cells to Fas-mediated apoptosis. Our results indicate that the induction of miR-128a expression by DNA demethylation is a novel mechanism of resistance to Fas-mediated apoptosis.

Prognostic impact of Fas-associated death domain, a key component in death receptor signaling, is dependent on the presence of lymph node metastasis in head and neck squamous cell carcinoma.

FAS-associated death domain (FADD) is a key adaptor protein that bridges a death receptor (e.g., death receptor 5; DR5) to caspase-8 to form the death-inducing signaling complex during apoptosis. The expression and prognostic impact of FADD in head and neck squamous cell carcinoma (HNSCC) have not been well studied. This study focuses on detecting FADD expression and analyzing its prognostic impact in primary and metastatic HNSCCs. We found a significant increase in FADD expression in primary tumors with lymph node metastasis (LNM) in comparison with primary tumors with no LNM. This increase was significantly less in the matched LNM tissues. Both univariate and multivariable analyses indicated that lower FADD expression was significantly associated with better disease-free survival and overall survival in HNSCC patients with LNM although FADD expression did not significantly affect survival of HNSCC patients without LNM . When combined with DR5 or caspase-8 expression, patients with LNM expressing both low FADD and DR5 or both low FADD and caspase-8 had significantly better prognosis than those expressing both high FADD and DR5 or both high FADD and caspase-8. However, the expression of both low FADD and caspase-8 was significantly linked to worse overall survival compared with both high FADD and caspase-8 expression in HNSCC patients without LNM. Hence, we suggest that FADD alone or together with DR5 and caspase-8 participates in metastatic process of HNSCC.

Molecules from apoptotic pathways modulate HIV-1 replication in Jurkat cells.

The replication of viruses involves control of some aspects of host cell homeostasis by modification of target cell metabolism and regulation of the apoptotic machinery. It is not well known whether molecules involved in apoptotic pathways affect human immunodeficiency virus type 1 (HIV-1) replication and regulate viral yields. Using the susceptible Jurkat cell line, we studied the relationship of apoptosis-associated molecules with HIV-1 virus production using a sensitive real-time RT-PCR assay. Here, we found that expression of proapoptotic proteins, including Fas ligand (FasL), FADD, or p53 significantly increased HIV-1 virus production. In contrast, the expression of antiapoptotic molecules, such as FLIP, Bcl-X(L), and XIAP, decreased HIV-1 virus production. Knockdown of Bax with siRNA and FADD with expression of its antisense mRNA also inhibited viral replication and the caspase-3 inhibitor, Z-DEVD, and decreased virus production. These data indicate that HIV-1 infection regulates the apoptosis process to facilitate viral replication and inhibition of apoptosis may inhibit HIV-1 replication and cytopathogenesis. We also discuss the effects of MAPK signaling pathways and apoptosis on HIV-1 replication.

High cell surface death receptor expression determines type I versus type II signaling.

Previous studies have suggested that there are two signaling pathways leading from ligation of the Fas receptor to induction of apoptosis. Type I signaling involves Fas ligand-induced recruitment of large amounts of FADD (FAS-associated death domain protein) and procaspase 8, leading to direct activation of caspase 3, whereas type II signaling involves Bid-mediated mitochondrial perturbation to amplify a more modest death receptor-initiated signal. The biochemical basis for this dichotomy has previously been unclear. Here we show that type I cells have a longer half-life for Fas message and express higher amounts of cell surface Fas, explaining the increased recruitment of FADD and subsequent signaling. Moreover, we demonstrate that cells with type II Fas signaling (Jurkat or HCT-15) can signal through a type I pathway upon forced receptor overexpression and that shRNA-mediated Fas down-regulation converts cells with type I signaling (A498) to type II signaling. Importantly, the same cells can exhibit type I signaling for Fas and type II signaling for TRAIL (TNF-α-related apoptosis-inducing ligand), indicating that the choice of signaling pathway is related to the specific receptor, not some other cellular feature. Additional experiments revealed that up-regulation of cell surface death receptor 5 levels by treatment with 7-ethyl-10-hydroxy-camptothecin converted TRAIL signaling in HCT116 cells from type II to type I. Collectively, these results suggest that the type I/type II dichotomy reflects differences in cell surface death receptor expression.

Necrostatin decreases oxidative damage, inflammation, and injury after neonatal HI.

Necrostatin-1 inhibits receptor-interacting protein (RIP)-1 kinase and programmed necrosis and is neuroprotective in adult rodent models. Owing to the prominence of necrosis and continuum cell death in neonatal hypoxia-ischemia (HI), we tested whether necrostatin was neuroprotective in the developing brain. Postnatal day (P)7 mice were exposed to HI and injected intracerebroventricularly with 0.1 µL of 80 µmol necrostatin, Nec-1, 5-(1H-Indol-3-ylmethyl)-(2-thio-3-methyl) hydantoin, or vehicle. Necrostatin significantly decreased injury in the forebrain and thalamus at P11 and P28. There was specific neuroprotection in necrostatin-treated males. Necrostatin treatment decreased necrotic cell death and increased apoptotic cell death. Hypoxia-ischemia enforced RIP1-RIP3 complex formation and inhibited RIP3-FADD (Fas-associated protein with death domain) interaction, and these effects were blocked by necrostatin. Necrostatin also decreased HI-induced oxidative damage to proteins and attenuated markers of inflammation coincidental with decreased nuclear factor-κB and caspase 1 activation, and FLIP ((Fas-associated death-domain-like IL-1ß-converting enzyme)-inhibitory protein) gene and protein expression. In this model of severe neonatal brain injury, we find that cellular necrosis can be managed therapeutically by a single dose of necrostatin, administered after HI, possibly by interrupting RIP1-RIP3-driven oxidative injury and inflammation. The effects of necrostatin treatment after HI reflect the importance of necrosis in the delayed phases of neonatal brain injury and represent a new direction for therapy of neonatal HI.

Hedgehog signaling drives cellular survival in human colon carcinoma cells.

Aberrant activation of Hedgehog (HH) signaling is implicated in many human cancers. Classical HH signaling is characterized by Smoothened (Smo)-dependent activation of Gli1 and Gli2, which transcriptionally regulate target genes. A small molecule inhibitor of Gli1 and Gli2, GANT61, was used to block HH signaling in human colon carcinoma cell lines that express HH signaling components. GANT61 administration induced robust cytotoxicity in 5 of 6 cell lines and moderate cytotoxicity in the remaining 1 cell line. In comparison, the classical Smo inhibitor, cyclopamine, induced modest cytotoxicity. Further, GANT61 treatment abolished the clonogenicity of all six human colon carcinoma cell lines. Analysis of the molecular mechanisms of GANT61-induced cytotoxicity in HT29 cells showed increased Fas expression and decreased expression of PDGFRα, which also regulates Fas. Furthermore, DR5 expression was increased whereas Bcl-2 (direct target of Gli2) was downregulated following GANT61 treatment. Suppression of Gli1 by shRNA mimicked the changes in gene expression observed in GANT61-treated cells. Overexpression of dominant-negative FADD (to abrogate Fas/DR5-mediated death receptor signaling) and/or Bcl-2 (to block mitochondria-mediated apoptosis) partially rescued GANT61-induced cytotoxicity in HT29 cells. Thus, activated GLI genes repress DR5 and Fas expressions while upregulating Bcl-2 and PDGFRα expressions to inhibit Fas and facilitate cell survival. Collectively, these results highlight the importance of Gli activation downstream of Smo as a therapeutic target in models of human colon carcinoma.